KR20120139462A - Method for producing cormus officinalis fermentation extract with increased antioxidative activity - Google Patents

Abstract

PURPOSE: A method for preparing Cormus officinalis fermentation extract with enhanced antioxidaiton effect is provided to increase loganin content and antioxidation effect and to effectively obtain the extract. CONSTITUTION: A method for preparing Cormus officinalis fermentation extract with enhanced antioxidation effect comprises: a step of washing Cormus officinalis and removing seeds; a step of drying and pulverizing Cormus officinalis; a step of inoculating Lactobacillus brevis(KCTC 13094) to MRS liquid medium and pre-culturing Lactobacillus brevis at 30-40 Deg.C. for 1-5 days; a step of steaming the pulverized Cormus officinalis and cooling; a step of inoculating the pre-cultured Lactobacillus brevis into steamed Cormus officinalis and fermenting; a step of adding purified water and performing hot water extraction at 100 Deg. C. for 1-3 hours; and a step of filtering and concentrating the extract by reduced pressure.

Description

Method for producing Corus of fermentation extract with increased antioxidant activity {Method for producing Cormus officinalis fermentation extract with increased antioxidative activity}

The present invention relates to a preparation method of cornus fermented extract having increased antioxidant activity.

Known extraction methods have been used to obtain the physiologically active ingredients contained in the herbal medicine. Known extraction methods include conventional water extraction method, alcohol extraction method, hot water extraction method, vapor pressure extraction method, etc. Recently, supercritical fluid extraction method, gamma irradiation extraction method, high pressure extraction method, high voltage pulse electric field (high A woltage pulsed electric field (PEF) extraction method, a microwave extraction method, and an ultrasonic extraction method are used.

The hydrothermal extraction method has low extraction efficiency, high energy consumption, and high heat, and has many disadvantages such as destruction of many physiologically active ingredients in herbal medicines, protein variation, soluble extraction, heat instability, and the like.

The ultrasonic extraction method is a form in which ultrasonic energy is converted into thermal energy from the inside rather than directly heating the heat, and is classified and used as a non-thermal treatment method by extracting using ultrasonic waves at a low temperature. It is not a specific heat treatment because it represents.

The high pressure extraction method is generally widely used as a method of extracting the pressure of 600 ~ 800 MPa, but because of the high pressure state, the price of the device is very expensive, there is a problem that the risk of handling the machine. In addition, another high pressure extraction method includes a supercritical fluid extraction method, which is a method for separating a specific essential oil component, and takes a long time to extract.

As described above, the supercritical fluid extraction method, gamma irradiation extraction method, high pressure extraction method, high woltage pulsed electric fields (PEF) extraction method, microwave extraction method, ultrasonic extraction method, etc. There are many limitations to its use and it does not show good advantages against the much higher investment costs than traditional extraction methods.

Therefore, a method of extracting the above extraction methods in parallel has been used. For example, the simultaneous treatment of the hydrothermal extraction method and the ultrasonic extraction method prevents the destruction of the physiologically active component by heat by the non-thermal treatment, and leads to the improvement of the extraction yield. However, the parallel extraction method is a disadvantage that is extracted by including a lot of other toxic substances contained in the herbal medicine.

Therefore, there is an urgent need for a research on a method of extracting a herbal medicine that can effectively extract a physiologically active ingredient in a herbal medicine.

Cornus officinalis, on the other hand, is a deciduous broad-leaved subtree belonging to the Cornaceae family. The cornus of dried fruit is dried and has a slightly warm, tasteful, sour and nonpoisonous nature. The fruit (fruit) contains glycosides such as cornin, morroniside, loganin, tannin, saponin, and organic acids such as wine, malic acid, tartaric acid, etc. It also contains vitamin A and a large amount of sugar. Seeds contain palmitic acid, oleic acid, linoleic acid and the like. Sansuyu makes the yin (왕) vigorous, Shinjeong (精) and Shingi (腎氣) to see and improve the sexual function, penis is enlarged and enlarged. In addition, it helps to cleanse (精髓) and warm your back and knees. Relieves frequent urination, swelling, nose stuffing, and deafness. In addition, it contains 15% or more of the saponin content is excellent in the convergence effect, and also has an effect of improving the skin by excellent harmful oxygen removal effect. In animal experiments, diuretic and blood pressure-lowering effects, arithmetic decoction water, staphylococcus aureus, inhibitory action of staphylococci, and inhibition of ascites cancer cells, hypoglycemic action, increase myocardial contractility, increase blood pressure, immunity Lymphatic cell proliferation is shown in the lineage, platelet aggregation inhibitory effect is also shown.

As described above, the cornus fruit contains a lot of various bioactive substances, but when extracting the bioactive substance from cornus milk using a conventional extraction method, very few bioactive substances as an active ingredient is obtained, the extraction efficiency is lowered There is this. Therefore, there is an urgent need for the development of an improved extraction method for effectively obtaining bioactive substances from cornus milk.

The present inventors studied the improved extraction method to effectively obtain a bioactive substance from cornus milk, steaming the cornus oil with steam and fermenting it with Lactobacillus brevis and then extracting the hydrothermal extract to obtain a cornus fermentation extract, the obtained cornus fermentation extract Loganin content contained in the higher than that in the cornus extract obtained by the conventional extraction method, it was confirmed that the antioxidant activity appeared better, and completed the present invention.

Therefore, the present invention is to provide a method for producing a cornus fermented extract with increased antioxidant activity.

The present invention

1) washing with cornus oil, removing seeds, drying and crushing the fruit,

2) Lactobacillus brevis (Lactobacillus brevis , KCTC 13094) inoculated in MRS (de Man, Rogosa, and Sharpe) liquid medium and pre-incubated,

3) steaming the pulverized cornus oil with steam and naturally cooling in a sealed state,

4) inoculating and fermenting precultured Lactobacillus brevis into steamed ground cornus;

5) adding purified water to the fermented cornus oil and extracting hot water, and

6) provides a method for producing a cornus fermentation extract with increased antioxidant activity, comprising the step of filtration under reduced pressure, concentration and drying under reduced pressure.

Hereinafter, the present invention will be described in detail.

The method for producing a cornus fermentation extract with increased antioxidant activity according to the present invention is characterized by steaming cornus oil with steam, fermenting it with Lactobacillus brevis and then extracting hot water.

Referring to the step-by-step detail of the preparation method of the cornus fermented extract of the present invention.

Step 1) is a step of drying and grinding the cornus oil, washing the cornus oil, removing the seeds, drying the fruit and pulverizing to a size of 0.2 ~ 0.5cm.

Wherein 2) is a step of pre-culture of lactic acid bacteria, Lactobacillus brevis (Lactobacillus brevis, KCTC 13094), the MRS (de Man, Rogosa, and Sharpe) before it for 1-5 days in inoculated to the liquid culture medium, and 30 ~ 40 ℃ Incubate.

Step 3) steaming the pulverized cornus oil with steam, steaming the dried pulverized cornus oil for 10-30 minutes, preferably 15 minutes at 110 ~ 130 ℃, preferably 121 ℃ and natural in a sealed state Cool.

Step 4) is a step of inoculating and fermenting the precultured lactobacillus brevis steamed crushed cornus, steamed Lactobacillus brevis steamed 3-10% (v / v), preferably 5 Inoculate at% (v / v) and ferment for 1-5 days at 30-40 ℃.

Step 5) is a step of extracting the fermented cornus oil by adding 5 to 10 times the amount of purified water to the fermented cornus oil after fermentation and extracting it for 1 to 3 hours at 100 ° C.

Step 6) is a step of obtaining a cornus fermentation extract in powder form. The cornus extract obtained in step 5) is filtered under reduced pressure, concentrated, and lyophilized to obtain a cornus fermentation extract in powder form.

As a result of analyzing the lignin content of the cornus fermented extract obtained by the above method by HPLC, the lignin content in the cornus fermented extract was 17.92 ± 0.07mg / g, and the cornus alcoholic extract (13.14 ± 0.15mg / g) It was higher than the cornus enzyme extract (14.58 ± 0.08mg / g) and the cornus enzyme hydrothermal extract (11.79 ± 0.11mg / g).

In addition, the DPPH radical scavenging activity, reducing power activity and hydrogen peroxide scavenging activity in the cornus fermented extract obtained by the above method are superior to the cornus alcohol extract, the cornus enzyme extract, and the cornus enzyme hydrothermal extract.

As described above, the extraction method according to the present invention by steaming the cornus milk with steam, fermented it with Lactobacillus brevis and then hot water extraction, it is possible to efficiently obtain a cornus milk fermentation extract having a high loganine content and increased antioxidant activity have. Therefore, the cornus fermented extract according to the present invention can be usefully used in the cosmetic composition for preventing skin aging.

The cornus fermented extract obtained by the extraction method according to the present invention has a higher loganine content and an excellent antioxidant activity than the cornus extract obtained by the conventional extraction method.

FIG. 1 is a diagram showing a calibration curve of loganin obtained by analyzing a standard solution of loganine diluted by concentration (0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5 mg / ml) by HPLC.
Figure 2 is a diagram showing the results of lignin analysis by HPLC analysis of each cornus extract.
Figure 3 is a diagram showing the DPPH radical scavenging activity according to the concentration of each cornus extract.
Figure 4 is a diagram showing the reducing power activity according to the concentration of each cornus extract.
5 is a diagram showing the hydrogen peroxide scavenging activity according to the concentration of each cornus extract.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  One  : Preparation of Cornus Fermented Extract

The cornus oil harvested from Shandong-myeon, Gurye-gun, Jeonnam was washed with water, the seeds were removed, and the fruits were dried and crushed into 0.2 ~ 0.5㎝ size. Lactobacillus brevis (KCTC 13094), a lactic acid bacterium, was inoculated into a 500 ml Erlenmeyer flask containing MRS (de Man, Rogosa, and Sharpe) liquid medium and pre-incubated in an incubator (HB-101M) for 72 hours at 37 ° C. It was. The dried ground cornus was steamed at 121 ° C. for 15 minutes and naturally cooled in sealed condition. Thereafter, the pre-cultured lactobacillus (Lactobacillus brevis) was put in a sterile spray container, inoculated by steaming evenly at 5% (v / v) of the weight of the steamed ground cornelic acid in steamed ground cornus, and then incubated in an incubator (HB-101M). Fermentation was carried out for 72 hours. After the fermentation was completed, 6 times the amount of purified water was added to the amount of cornus milk and extracted at 100 ° C. for 1 hour using an extractor (KSNP B1130). A paper filter (ADVANTEC, 110 mm) having a membrane pore size of 5 μm was placed on the filter portion, and about 3 cm of celite was added thereto. The extract was filtered under reduced pressure. The filtrate was concentrated under reduced pressure using a rotary vacuum evaporator (EYELA, N-1000) at a temperature of 700 mmHg and a temperature of 50 to 60 ° C. until the final solid content was 50%. Thereafter, the concentrate was frozen in an ultra-low temperature freezer at -70 ° C, and then dried with a lyophilizer (EYELA, FDU-2100) to prepare a cornus fermentation extract in powder form.

Comparative example  One  Cornus Alcohol  Preparation of extract

500 ml of 70% ethanol was added to 300 g of dried cornus oil, and the extract was repeatedly extracted three times at 70 ° C. for 1 hour using a reflux cooling extractor. The extract was filtered under reduced pressure, concentrated under reduced pressure until the final solids content of the filtrate was 50%, and then lyophilized to prepare a cornus ethanol extract in powder form.

Comparative example  2  : Preparation of Cornus Enzyme Extract

2,400 ml of purified water was added to 400 g of dry cornus, and pectinase (Pectinex 5XL, NOVOZYMES) and a complex enzyme (Viscozyme L, Biosis) were each added 0.65% (v / v) to the weight of dry cornus. After 4 hours 30 minutes at 200rpm, 50 ℃, pH 5.0 using a stirrer. Then, inactivated for 15 minutes at 100 ℃ to stop the activity of the enzyme. The extract was filtered under reduced pressure, concentrated under reduced pressure until the final solids content of the filtrate was 50%, and then lyophilized to prepare an extract of cornus enzyme in powder form.

Comparative example  3  Cornus enzyme Heat number  Preparation of extract

2,400 ml of purified water was added to 400 g of dry cornus, and pectinase (Pectinex 5XL, NOVOZYMES) and a complex enzyme (Viscozyme L, Biosis) were each added 0.65% (v / v) to the weight of dry cornus. After 4 hours 30 minutes at 200rpm, 50 ℃, pH 5.0 using a stirrer. Then, the reaction solution was naturally cooled and extracted with the extractor (KSNP B1130) at 100 ° C. for 1 hour. The extract was filtered under reduced pressure, concentrated under reduced pressure until the final solid content of the filtrate was 50%, and then lyophilized to prepare a hydrous enzyme hydrothermal extract in powder form.

Experimental Example  One  : HPLC  Of each cornus extract by analysis Loganine  Quantitative analysis

For quantitative analysis on the sample solution of cornus extract, the lignin content was analyzed using HPLC. The standard solution was used to completely dissolve the loganin (loganin) in methanol and filtered through a 0.45㎛ syringe filter (ADVENTEC), each of the cornus extract was dissolved in ethanol and filtered by 0.45㎛ syringe filter and quantitatively analyzed. HPLC analysis conditions are shown in Table 1. A quantitative analysis is performed by creating a calibration curve for the peak area ratio and the concentration of the standard solution for each loganine obtained by diluting the loganine to a concentration of 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5 mg / ml. It was.

The calibration curve of loganan obtained by HPLC analysis of ligannine standard solution diluted by concentration (0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.5 mg / ml) is shown in FIG. Loganin quantitative analysis of each cornus extract is shown in Figure 2 and Table 2.

HPLC analysis conditions device( Agilent Technologies ) Pump Quat Pump (G1311A) Detector VWD (G1314B) Auto sampler ALS (G1329A) column Gemini 5u C ₁₈ (110A 4.6 × 250 mm) Operating conditions UV wavelength 203 nm Column temperature 35 ℃ Dose 10 μl Flow rate 1.0ml / min Mobile phase A Distilled water Mobile phase B Acetonitrile gradient( Gradient profile ) Time (min) % A % B Flow rate (ml / min) 0 95 5 1.0 20 70 30 30 70 30 35 20 80

Loganin Content of Cornus Extracts by HPLC Analysis Cornus Extract Loganine Content (mg / g) Cornus Fermented Extract (Example 1) 17.92 ± 0.07 Cornus alcohol extract (Comparative Example 1) 13.14 ± 0.15 Cornus enzyme extract (Comparative Example 2) 14.58 ± 0.08 Cornus enzyme hydrothermal extract (Comparative Example 3) 11.79 ± 0.11

As shown in FIG. 1, FIG. 2, and Table 2, the correlation coefficient (R ² ) of the calibration curve of loganin was 0.999, indicating a high linearity, and the loganine content of each cornus extract was 17.92 in the cornus fermented extract. The highest value was ± 0.07 mg / g.

Experimental Example  2  : Determination of Antioxidant Activity of Cornus Extract

In order to confirm the antioxidant activity of each cornus extract, DPPH radical scavenging activity, reducing power activity, hydrogen peroxide scavenging activity was measured.

One. DPPH Radical  Scavenging activity measurement

The test method of Brand-Williams (1995) was modified to determine the scavenging ability of Cornus extract against DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals. Specifically, the cornus extracts prepared in Example 1 and Comparative Examples 1 to 3 were added to the DPPH solution by concentration (10, 50, 100, 500, 1000 ㎍ / ml) and mixed well for 10 seconds. Then, the mixed solution was reacted at room temperature for 1 hour, and then absorbance was measured at 540 nm. DPPH radical scavenging measurement was expressed by converting the absorbance ratio of the sample (Hansu extract) and the control group into% values. As a positive control group, vitamin C, which is well known for its antioxidant activity, was used.

The results are shown in FIG.

As shown in Figure 3, it was confirmed that the DPPH radical scavenging activity was excellent as the concentration increased in all cornus extract. Cornus enzyme extract and cornus fermented extract showed similar DPPH radical scavenging activity at the concentration of 50 µg / ml or more, but the concentration of DPPH radical scavenging activity was highest in the cornus fermentation extract at the concentration of 100 µg / ml.

2. Measurement of reducing power activity

1 ml of 200 mM phosphate buffer solution of pH 6.6 and 1% potassium ferricyanide were sequentially added to each 1 ml of the cornus oil extract prepared in Examples 1 and Comparative Examples 1 to 3, followed by stirring. The reaction was carried out for 20 minutes in a water bath of. 1 ml of 10% trichloroacetic acid (TCA) solution was added thereto, and centrifuged at 13,500 × g for 15 minutes. 1 ml of each supernatant was mixed with 1 ml of distilled water and ferrous chloride, and the absorbance was measured at 700 nm. Reducing power of each cornus extract was converted into% value of the absorbance ratio of the sample (cornus extract) addition group and the control group.

The results are shown in Fig.

As shown in Figure 4, each cornus extract showed a similar reducing activity at a concentration of 10 ~ 100㎍ / ㎖, but at a concentration of 500㎍ / ㎖ or more fermented extract of cornus> Cornus enzyme extract> Cornus enzyme hydrothermal extract> Cornus alcohol extract It showed reducing power activity in that order. In particular, the reducing activity of the cornus fermented extract showed higher activity than that of vitamin C, a positive control group.

3. Determination of hydrogen peroxide scavenging activity

80 µl each of the Cornus extract prepared in Example 1 and Comparative Examples 1 to 3, 20 µl of 10 mM hydrogen peroxide and 100 µl of 0.01 M phosphate buffer solution (pH 5.0) were reacted at 37 ° C. for 5 minutes. Thereafter, 15 µl of 1.25 mM ABTS (2,2'-aziono-bis (3-ethylbenzthiazoline-6-sulfonic acid) and 30 µl of peroxidase were reacted at 37 ° C for 10 minutes to measure absorbance at 405 nm. The hydrogen peroxide scavenging activity was calculated by converting the absorbance ratio before and after the addition of the sample (cortex extract) into a% value.

The results are shown in Fig.

As shown in FIG. 5, the hydrogen peroxide scavenging activity of each cornus extract was not significantly different from the negative control group at a concentration of 10 μg / ml, but was higher than that of the negative control group, and showed the highest hydrogen peroxide scavenging activity in the cornus fermentation extract. It was.

Claims (6)

1) washing with cornus oil, removing seeds, drying and crushing the fruit,
2) Lactobacillus brevis (Lactobacillus brevis , KCTC 13094) inoculated in MRS (de Man, Rogosa, and Sharpe) liquid medium and pre-incubated,
3) steaming the pulverized cornus oil with steam and naturally cooling in a sealed state,
4) inoculating and fermenting precultured Lactobacillus brevis into steamed ground cornus;
5) adding purified water to the fermented cornus oil and extracting hot water, and
6) filtration process of the extract under reduced pressure, concentration and drying, a method for producing a cornus fermentation extract with increased antioxidant activity. The method of claim 1, wherein the pre-culture in step 2) is performed for 1 to 5 days at 30 to 40 ° C. The method of claim 1, wherein the cornus pulverized in step 3) is steamed at 110 to 130 ° C for 10 to 30 minutes, producing a cornus fermented extract having increased antioxidant activity. According to claim 1, Lactobacillus brevis pre-cultured in step 4) is inoculated at 3 to 10% (v / v) relative to the weight of steamed ground cornelic acid, cornus fermentation extract with increased antioxidant activity Manufacturing method. The method of claim 1, wherein the fermentation in step 4) is performed at 30 to 40 ° C. for 1 to 5 days. According to claim 1, wherein the hot water extraction in step 5) is added to the fermented cornus milk 5 to 10 times the amount of purified water and the extract is for 1 to 3 hours at 100 ℃, cornus milk with increased antioxidant activity Method for preparing fermented extract.

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